An 80-year-old injection solution of ouabain was analyzed using HPLC-MS. A new separation method involving hydrophilic interaction liquid chromatography (HILIC) using an amide-based stationary phase with mass spectrometric detection was developed for the analysis.
The optimized mobile phase composition was acetonitrile:water 85:15 (v/v) at the flow rate of 0.2 cm(3)/min under the isocratic elution. The ouabain content was found reduced to 56% of declared content and probable degradation product with oxidized hydroxy group was established.
The ESI-MS2 fragmentation mechanism of ouabain was proposed.